Nonspecific probe binding and automatic gating in flow cytometry and fluorescence activated cell sorting (FACS)

Area of Science:

• Cell biology
• Biophysics
• Biochemistry

Background:

• Flow cytometry is crucial for distinguishing mutant from wild-type cells using fluorescent probes.
• Non-specific probe binding to cell membranes can lead to false positives and inaccurate cell quantification.
• Existing methods for analyzing flow cytometry data may not fully account for these binding complexities.

Purpose of the Study:

• To develop a kinetic model for fluorescent probe binding dynamics in flow cytometry.
• To analyze the impact of non-specific binding on assay accuracy.
• To introduce improved methods for quantifying mutant cells and inferring experimental parameters.

Main Methods:

• Derivation and analysis of a kinetic model for probe binding.
• Application of a two-species Langmuir adsorption model.
• Analytical derivation of an expectation maximization method for cell quantification.

Main Results:

• The kinetic model effectively analyzes non-specific binding effects.
• An expectation maximization method provides accurate mutant cell estimates.
• A novel method is proposed for inferring physical and experimental parameters.

Conclusions:

• The developed model enhances quantitative analysis of flow cytometry data.
• Improved accuracy in mutant cell identification and parameter estimation is achieved.
• This work offers a more robust approach to flow cytometry data interpretation.